Transgenic animals with customizable traits

ABSTRACT

Disclosed are materials and methods for creating customizable traits in animals. In the demonstration of the principle of the subject invention, a keratin-14 specific promoter is used with, red fluorescent protein in the loxp cassette, dominant black (ΔG23) beta defensin 103 in the pigment cassette, and an SV40 (with intron) polyadenylation sequence. When Cre recombinase (or HTNCre) is applied to the animal&#39;s skin in a carrier base (e.g., lipid bilayers), fur is permanently genetically modified to turn black in the shape in which it was applied.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the priority benefit of U.S. ProvisionalApplication Ser. No. 61/598,987, filed Feb. 15, 2012, which isincorporated herein by reference in its entirety.

The Sequence Listing for this application is labeledSeqList-15Feb13_ST25.txt which was created on Feb. 15, 2013 and is 66KB. The entire content of the sequence listing is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

This invention relates to genetic modification of animals in order toeffect controlled changes in specified traits such as, for example,changes in skin and/or fur pigmentation.

BACKGROUND OF THE INVENTION

The only pigment synthesized in most animal species, and the onlypigment at all in mammalian species, is melanin. There are two classesof melanin pheomelanin, which produces a blond or red color, andeumelanin, which produces a dark brown or black color. Both classes ofmelanin are synthesized from tyrosine, but their synthetic pathwaysdiverge after production of dopaquinone.

The primary switch controlling whether a particular melanocyte producespheomelanin or eumelanin is the melanocortin receptor (MC1R). MC1Rpolymorphisms also appear to be the primary determinant of red or blondpheomelanin.

The melanocortin receptor can be activated by any of themelanocyte-stimulating hormones (MSH), most commonly by α-MSH, but alsoby β-defensin 103. Conversely, activation of MC1R can be inhibitedthrough expression of the agouti signaling protein (ASP). The β-defensin103 signal is dominant over the ASP signal and the ASP signal isdominant over the MSH signal. Further, there are mutations andpolymorphisms in all of these genes that increase or decrease theiractivity. Coat color is further modulated by multiple modulators ofmelanin production and transport, including tyrosinase (TYR), thetyrosinase transporter OCA2, and the ubiquitination gene HERC2, amongothers.

Plants make multiple additional classes of pigments, includingchlorophyll, carotenoids, anthrocyanins, and betalains. Of thesepigments, carotenoids can be most easily transferred to animals becauseproduction of carotenoid dyes in plants relies on a precursor that canalso be found in animals. Geranylgeranyl pyrophosphate is anintermediate in the HMG-CoA reductase pathway, and is used as aprecursor for synthesizing steroids and sterols. With the addition of4-5 plant proteins, geranylgeranyl pyrophosphate can instead be used asa precursor for synthesizing carotene yellow and orange, or torulenered. The transfer of plant pigments to animals has occurred in nature:the aphid A. pisum has several genes somehow transferred from fungi.Carotenoids have also been produced in genetically-engineered yeast,which belong to the animal kingdom.

The only modification of surface pigment ever attempted by geneticintervention in multicellular animals is the wholesale change of animalsfrom light to dark or dark to light. No more detailed patterns have beencreated. Carotene dyes have never been naturally found in animals morecomplicated than aphids, and have never been engineered into anymulticellular animal. Before the present invention, customizable coloror patterns in the skin or fur of animal species have not been created.

BRIEF SUMMARY

The subject invention provides materials and methods for creatingcustomizable traits in animals. For example, the subject inventionprovides materials and methods for creating customizable patterns in theskin and/or fur of animals. In other embodiments, the customizable traitcan involve the length and/or texture of animal skin or fur.Alternatively, the subject invention can be used to effect controlledchanges in the texture, structural strength, and/or length of animalnail, claw, and/or horn.

In one specific embodiment, the methods of the subject inventioncomprise indtroducing into the cells of an animal a genetic constructcomprising a keratin-14 specific promoter, red fluorescent protein in aloxp cassette, dominant black (ΔG23) beta defensin 103 in a pigmentcassette, and an SV40 (with intron) polyadenylation sequence. When acomposition comprising Cre recombinase (or HTNCre) is then applied tothe skin of an animal having the genetic construct, the fur of theanimal will turn black where the composition was applied. In this way,the fur is permanently genetically modified to turn color in a desiredshape.

Thus, in one embodiment the subject invention provides a method ofcreating customizable permanent patterns in the skin and/or fur ofanimals.

In another embodiment the subject invention provides methods forproducing multicolor patterns in the skin and/or fur of animal species.

In a further embodiment the subject invention provides a method ofcreating customizable patterns in the skin and/or fur of animal speciessuch that the animal would continue to grow fur to sustain those colorsthroughout its lifetime.

The invention also provides methods of creating customizable predefinedpatterns of stripes that are heritable within that animal.

In one embodiment, the present invention provides transdermalapplication of one or more activating factors to drive recombination andpermanent transgene expression in the transgenic animals of the presentinvention. In certain specific embodiments, the activating factorsuseful according to the present invention include, but are not limitedto, recombinase proteins, small molecules (such as, doxycycline, cumate,ecdysone, etc) capable of inducing the expression of recombinase,viruses that capable of inducing the expression of recombinase, andnucleic acid (such as DNA) constructs that drive the expression ofrecombinase.

In certain embodiments, the activating factor is applied to the surfaceof the animal skin, either alone or in a carrier solution (e.g.,liposomes, solvents, mixtures containing DMSO, etc.). In one embodiment,the activating factor is applied intradermally (such as with the use ofa tattoo needle) or subdermally.

In one embodiment, the transgenic animal comprises one or more exogenousnucleic acid molecules including, but not limited to,pigmentation-related genes, coat/hair quality genes (such as genes forcontrolling the length and/or curliness of animal hair), genes relatedto nail/claw or horn quality (such as a nucleic acid molecule encodingcross-linking keratin), and genes for synthesis and/or expression ofplant pigments in animal cells.

In certain embodiments, promoters useful according to the presentinvention include, but are not limited to, skin-specific promoters(e.g., keratin specific promoter), melanocyte specific promoters (e.g.,MCR promoter), constitutive promoters (e.g., beta-globin promoter, CMVpromoter), and promoters responsive to circulating factors such asNF-kn, interferon gamma estrogen, and glucocorticoids.

In certain embodiments, the present invention provides the use ofmultiple types of recombinase targets to allow specific activation ofdifferent genes selectively through application of differentrecombinases. In one embodiment, multiple recombinase targets are usedto allow multiple colors to be created after birth of the animal.

In one embodiment, in a transgenic dog with a naturally golden fur, Crerecombinase activates the production of dog fur with black color, andFlp recombinase activates the production of dog fur with red color.

In one embodiment, the present invention provides the use of nativepromoters to drive coat pigmentation without the need for an externalactivating factor. In a specific embodiment, the native promoter relatesto defining somite boundaries in animal development.

In one embodiment, the native heterologous promoter is used to createcoat patterns in a different species, such as, for example, using theTabby or Ticked promoters found in cats to drive coat coloration indogs.

In certain embodiments, the present invention provides geneticallymodified animals with coat patterns that are permanently customizableafter birth.

In certain embodiments, the present invention provides geneticallymodified animals with coat colors not normally found in mammals.

In certain embodiments, the present invention provides geneticallymodified cattle, sheep or other animals that have permanentidentification marks (such as, a number or a bar code) growing in theircoat.

In certain embodiments, the present invention provides geneticallymodified cattle, sheep or other animals that have “invisible” marks intheir coat that can change color in response to changes in health orphysiological conditions.

In one embodiment, the transgenic cattle or sheep or other animals canexpress one symbol on the coat or fur, wherein that symbol can changecolor if the animal has chronic activation of NF-kB, and another symbolthat can change color if the animal has chronic activation ofinterferon-gamma.

In certain embodiments, the present invention provides geneticallymodified animals born with coat colors and patterns not normally foundin their native species.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic depiction of an embodiment of a genetic constructfor effecting color change in animals.

FIG. 2 is a schematic depiction of an embodiment of a genetic constructfor creating customizable patterns and color in animal skin or fur.

FIG. 3 is a schematic depiction of an embodiment of genetic constructsfor creating customizable patterns and color in animal skin or fur.

FIG. 4 is a schematic depiction of one embodiment of a genetic constructfor creating customizable patterns and color in the skin or fur of mice.The construct comprises a Keratin14 promoter, which is expressed in allskin fibroblasts, to drive the dominant black form of signaling moleculeβ-Def103. The expression of β-Def103 is blocked by a LoxP excisablenucleic acid encoding ring finger protein (RFP), which is used as amarker. The nucleic acid molecule encoding RFP is not required for theconstruct, and can be replaced by STOPs.

FIG. 5 (A) shows that beta-Def103 expression activated by transdermalapplication of recombinase creates a genetically permanent alteration inthe coat or fur of an adult mouse. (B) Fine control of markingalteration can be achieved: a mouse with a single narrow black line iscreated by injection of recombinase. The change in skin/fur pattern haspersisted through multiple cycles of coat regrowth, indicatingsuccessful alteration of resident stem cells.

FIG. 6 shows that coat color of the transgenic mice can be titratedthrough the amount of recombinase applied to the mouse skin. Increasingthe amounts of recombinase applied to the mice increases the level oftransgene expression and eumelanin activation.

FIG. 7 is a schematic depiction of one embodiment of a genetic constructfor knock-in cattle with spermatozoa-specific expression of enhancedgreen fluorescent protein (EGFP), and recombinase-mediatedmelanocyte-specific expression of a dominant negative Rab7. A nucleicacid sequence encoding neomycin resistance biomarker protein, which canbe excised through PIGGYBAC™ transposons, is placed in the center of theconstruct, and the construct is flanked by short homology arms.

FIG. 8 shows a depiction of a Black Angus heifer genetically engineeredto express a customizable identification pattern in the skin.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the amino acid sequence of a bifunctional enzymeCarRP-like isoform 1 [Acyrthosiphon pisum] (GenBank Accession No.XP_(—)001943170).

SEQ ID NO:2 is the amino acid sequence of a bifunctional enzymeCarRP-like [Acyrthosiphon pisum] (GenBank Accession No.XP_(—)001950787).

SEQ ID NO:3 is the amino acid sequence of a lycopene cyclase/phytoenesynthase-like [Acyrthosiphon pisum] (GenBank Accession No.XP_(—)001950868).

SEQ ID NO:4 is the amino acid sequence of a phytoene dehydrogenase-like[Acyrthosiphon pisum] (GenBank Accession No. XP_(—)001943225).

SEQ ID NO:5 is the amino acid sequence of a phytoene dehydrogenase-like[Acyrthosiphon pisum] (GenBank Accession No. XP_(—)001950764).

SEQ ID NO:6 is the amino acid sequence of a phytoene dehydrogenase-like[Acyrthosiphon pisum] (Genbank Accession No. XP_(—)001946689).

SEQ ID NO:7 is the amino acid sequence of a phytoene dehydrogenase-like[Acyrthosiphon pisum] (Genbank Accession No. XP_(—)001943938).

SEQ ID NO:8 is the amino acid sequence of a melanocortin 1 receptor(GenBank Accession No. EDL11741).

SEQ ID NO:9 is the amino acid sequence of an alpha melanocytestimulating honnone (MSH).

SEQ ID NO:10 is the amino acid sequence of a beta melanocyte stimulatinghormone (MSH).

SEQ ID NO:11 is the amino acid sequence of a beta melanocyte stimulatinghormone (MSH).

SEQ ID NO:12 is the amino acid sequence of a gamma melanocytestimulating hormone (MSH).

SEQ ID NO:13 is the amino acid sequence of a (3-defensin protein(GenBank Accession No. AAT67592).

SEQ ID NO:14 is the amino acid sequence of an agouti signaling proteinprecursor (GenBank Accession No. NP_(—)056585s).

SEQ ID NO:15 is the amino acid sequence of a tyrosinase (TYR) (GenBankAccession No. BAA00341).

SEQ ID NO:16 is the amino acid sequence of a melanocyte-specifictransporter protein (GenBank Accession No. Q62052).

SEQ ID NO:17 is the amino acid sequence of a rab proteingeranylgeranyltransferase component A2 (GenBank Accession No.NP_(—)067325).

SEQ ID NO:18 is the amino acid sequence of a ras-related protein Rab-7a(GenBank Accession No. NP_(—)033031).

SEQ ID NO:19 is the amino acid sequence of a probable E3ubiquitin-protein ligase (HERC2) (GenBank Accession No. NP_(—)084390).

DETAILED DISCLOSURE

The subject invention provides materials and methods for creatingcustomizable traits in animals. For example, the subject inventionprovides materials and methods for creating customizable patterns in theskin and/or fur of animals. In other embodiments, the customizable traitcan involve the length and/or texture of animal skin or fur.Alternatively, the subject invention can be used to effect controlledchanges in the texture, structural strength, and/or length of animalhair, nail, claw and/or horn.

In one specific embodiment, the methods of the subject inventioncomprise introducing into the cells of an animal a genetic constructcomprising a keratin-14 specific promoter, red fluorescent protein in aloxp cassette, dominant black (ΔG23) beta defensin 103 in a pigmentcassette, and an SV40 (with intron) polyadenylation sequence. When acomposition comprising Cre recombinase (or HTNCre) is then applied tothe skin of an animal having the genetic construct, the fur of theanimal turns black where the composition is applied. In this way, thefur is permanently genetically modified to turn color in a desiredshape.

Thus, in one embodiment the subject invention provides a method ofcreating customizable permanent patterns in the skin and/or fur ofanimals.

In another embodiment the subject invention provides methods forproducing multicolor patterns in the skin and/or fur of animal species.

In a further embodiment the subject invention provides a method ofcreating customizable patterns in the skin and/or fur of animal speciessuch that the animal would continue to grow fur to sustain those colorsthroughout its lifetime.

The invention also provides methods of creating customizable predefinedpatterns of stripes that are heritable within that animal.

In one embodiment, the transgenic animal has heritable soft claws orsoft hair or fur.

In another embodiment, the present invention provides cells, tissues, orparts (such as skin, hair, fur) of the transgenic animal, and usesthereof. In one embodiment, the transgenic animal has customizable colorand/or patterns in the skin and/or fur, and the skin or fur can besubsequently removed from the transgenic animal for production of hides,fur, leather, etc, useful for production of clothing, rugs, shoes, horsetack, horse harness, upholstery, and other leather goods.

Transgenic Animals with Customizable Traits

In one embodiment, the present invention provides a transgenic animalwith customizable traits, wherein the transgenic animal (such as in itsgenome) comprises:

an exogenous nucleic acid molecule encoding a protein of interest,wherein the nucleic acid molecule is operably linked to a promoter andis under the control of an inducible gene expression system thatrequires the presence of an inducing agent to activate gene expression;

wherein the expression of the exogenous nucleic acid molecule isinhibited in the absence of the inducing agent.

In certain embodiments, the exogenous nucleic acid molecule that encodesa protein is selected from pigment proteins; proteins involved in thesynthesis and/or transport of pigments; luminescent (such asfluorescent) proteins; proteins involving the length and/or texture ofanimal skin or fur; and proteins involved in the texture, structuralstrength, and/or length of animal nail, claw, and/or horn.

In one specific embodiment, the present invention provides a transgenicanimal with customizable fur or skin pigmentation, wherein the genome ofthe transgenic animal comprises:

an exogenous nucleic acid molecule encoding a pigment protein ofinterest or a protein involved in the synthesis and/or transport of apigment of interest, wherein the exogenous nucleic acid molecule isoperably linked to a promoter and is under the control of an induciblegene expression system that is a site-specific recombination system,

wherein the site-specific recombination system inhibits the expressionof the first nucleic acid molecule in the absence of site-specificrecombination.

The expression of the exogenous nucleic acid molecule can be inducedafter the application of, for example, a recombinase to the transgenicanimal.

In one embodiment, the exogenous nucleic acid molecule, the promoter,and/or the site-specific recombination system are contained in apigmentation construct.

In one embodiment, the genome of the transgenic animal comprises anexogenous nucleic acid molecule whose expression is under the control ofa Cre/LoxP recombination system, wherein the Cre/LoxP recombinationsystem prevents the expression of the exogenous nucleic acid molecule.In one specific embodiment, the Cre/LoxP recombination system comprisesa lox-stop-lox (LSL) sequence.

In one embodiment, the pigmentation construct is transferred into cells,such as fertilized ova. The pigmentation construct can be transferredinto fertilized ova using any conventional means, including, but notlimited to, lentivirii, pronuclear injections, and intracytoplasmicsperm injection (ICSI).

In certain embodiments, one or more pigmentation constructs areintroduced into the genome of the transgenic animal. The pigmentationconstruct can comprise more than one exogenous nucleic acid molecule,each nucleic acid molecule encoding a protein of interest.

In certain embodiments, the genome of the transgenic animal comprisesmore than one inducible gene expression systems to control theexpression of the nucleic acid molecules of interest.

In one specific embodiment, the present invention provides a transgenicanimal with customizable traits (such as fur or skin pigmentation),wherein the genome of the transgenic animal comprises:

a first exogenous nucleic acid molecule encoding a protein of interest(such as a pigmentation protein) operably linked to a first promoter andunder the control of a loxP site, wherein the loxP site prevents theexpression of the first exogenous nucleic acid molecule in the absenceof Cre recombinase protein; and

a second nucleic acid molecule encoding a Cre recombinase protein,operably linked to a second promoter.

In another specific embodiment, the present invention provides atransgenic animal with customizable traits (such as fur or skinpigmentation), wherein the genome of the transgenic animal comprises:

a first exogenous nucleic acid molecule encoding a protein of interest(such as pigmentation protein) operably linked to a first promoter andunder the control of a loxP site, wherein the loxP site prevents theexpression of the first nucleic acid molecule in the absence of Crerecombinase protein;

a second nucleic acid encoding a reverse tRA (rtTA), operably linked toa second promoter; and

a third nucleic acid molecule encoding a Cre recombinase protein,operably linked to a third promoter under the control of a TetOoperator.

FIGS. 2 and 3 show an embodiment of the expression constructs, whereinthe expression of the exogenous nucleic acid molecule of interest (suchas pigment proteins and proteins involved in the synthesis of biologicalpigments) is under the control of the Cre-LoxP recombination system anda tetracycline (Tet)-controlled transcription activation system.

In one specific embodiment, doxycycline (or ecdysone, etc) mixed withDMSO carrier is applied to a transgenic animal with a gold fur color,whereby the color of the transgenic animal turns red; subsequently, Creor HTNCre mixed with DMSO is applied to the transgenic animal to producea black color.

In another specific embodiment, the genome of the transgenic animalcomprises an exogenous nucleic acid molecule the expression of which isunder the control of a tetracycline (Tet)-controlled transcriptionalactivation system.

In another embodiment, the present invention provides a transgenicanimal with customizable traits (such as fur or skin pigmentation),wherein the genome of the transgenic animal comprises: a first exogenousnucleic acid molecule encoding a protein of interest (such as apigmentation protein) operably linked to a first promoter and under thecontrol of an inducible gene expression system (e.g., a tetracycline(Tet)-controlled transcriptional activation system), wherein theinducible gene expression system, in its inactivated state (absent ofinduction), prevents the expression of the first nucleic acid molecule.

The transgenic animal can be of any species, including, but not limitedto, mammalian species including, but not limited to, domesticated andlaboratory animals such as dogs, cats, mice, rats, guinea pigs, andhamsters; livestock such as horses, cattle, pigs, sheep, goats, ducks,geese, and chickens; primates such as apes, chimpanzees, orangutans,humans, and monkeys; fish; amphibians such as frogs and salamanders;reptiles such as snakes and lizards; and other animals such as fox,weasels, rabbits, mink, beavers, ermines, otters, sable, seals, coyotes,chinchillas, deer, muskrats, and possum. In certain embodiments, theanimal is not a human.

Pigment Proteins

The term “pigment protein,” as used herein, refers to a proteincomprising a pigment. The term “pigment,” as used herein, refers to amaterial that does not emit light but changes the color of reflected ortransmitted light as the result of wavelength-selective absorption; thisphysical process differs from fluorescence, phosphorescence, and otherforms of luminescence, in which a material emits light. Pigment proteinsinclude, but are not limited to, chromoproteins such as cytochromes andflavoproteins.

Luminescent Proteins

The term “luminescent protein,” as used herein, refers to a protein thatemits light. Luminescent proteins useful according to the presentinvention include, but are not limited to, fluorescent proteinsincluding, but not limited to, green fluorescent protein, yellowfluorescent protein, cyan fluorescent protein, and red fluorescentprotein; and phosphorescent proteins. Fluorescent proteins are membersof a class of proteins that share the unique property of beingself-sufficient to form a visible wavelength chromophore from a sequenceof three amino acids within their own polypeptide sequence. A variety ofluminescent proteins, including fluorescent proteins, are publiclyknown. Fluorescent proteins useful according to the present inventioninclude, but are not limited w, the fluorescent proteins disclosed inU.S. Pat. No. 7,160,698, U.S. Application Publication Nos. 2009/0221799,2009/0092960, 2007/0204355, 2007/0122851, 2006/0183133, 2005/0048609,2012/0238726, 2012/0034643, 2011/0269945, 2011/0223636, 2011/0152502,2011/0126305, 2011/0099646, 2010/0286370, 2010/0233726, 2010/0184116,2010/0087006, 2010/0035287, 2007/0021598, 2005/0244921, 2005/0221338,2004/0146972, and 2001/0003650, all of which are hereby incorporated byreference in their entireties.

Proteins Involved in the Synthesis of Biological Pigments

Proteins involved in the synthesis of biological pigments include, butare not limited to, the wild-type or mutant forms of melanocortinreceptor (MC1R), melanocyte stimulating hormones (MSH) (e.g., α-MSH,β-MSH, γ-MSH), β-defensin 103, agouti signaling protein (ASP),tyrosinase (TYR), melanocyte-specific transporter protein, Ras-relatedprotein Rab-7, rab protein geranylgeranyltransferase component A2, andprobable E3 ubiquitin-protein ligase (HERC2).

In certain embodiments, the genome of the transgenic animal comprises anexogenous nucleic acid molecule encoding a protein involved in thesynthesis of a biological pigment.

Nucleic acid molecules encoding proteins involved in the synthesisand/or transport of biological pigments can be derived from genesincluding, but not limited to, the dominant MC E92K and the agouti gene.In certain embodiments, the genome of the transgenic animal comprises anucleic acid molecule encoding a protein involved in the synthesisand/or transport of biological pigments including, but not limited to,melanins (e.g., pheomelanin, eumelanin); urochrome; chlorophyll;bilirubin; biliverdin; phycobilin; phycoerythrobilin; stercobilin;urobilin; hemocyanin; hemoglobin; myoglobin; luciferins; carotenoids,including hematochromes, carotenes (e.g., alpha and beta carotene,lycopene, rhodopsin), xanthophylls (e.g., canthaxanthin, zeaxanthin,lutein); phytochrome; phycobiliproteins (e.g., R-phycoerythrin (R-PE),B-phycoerythrin (B-PE), C-phycocyanin (CPC), allophycocyanin (APC));polyene enolates; and flavonoids.

Carotenoid pigments in yellow, red, or orange can be synthesized inanimals that express phytoenesynthases, desaturases, and cyclases, asdescribed in Moran (2010) and Verdoes (2003). In one embodiment, thecarotenoid dyes are synthesized using geranylgeranyl pyrophosphate as asubstrate.

Proteins involved in the synthesis and/or transport of biologicalpigments include, but are not limited to, bifunctional enzyme CarRP-likeisoform 1 [Acyrthosiphon pisum] (such as, GenBank Accession No.XP_(—)001943170 (SEQ ID NO:1)), bifunctional enzyme CarRP-like[Acyrthosiphon pisum] (such as, GenBank Accession No. XP_(—)001950787(SEQ ID NO:2)), lycopene cyclase/phytoene synthase-like [Acyrthosiphonpisum] (such as GenBank Accession No. XP_(—)001950868 (SEQ ID NO:3)),phytoene dehydrogenase-like [Acyrthosiphon pisum] (such as, GenBankAccession No. XP_(—)001943225 (SEQ ID NO:4)), phytoenedehydrogenase-like [Acyrthosiphon pisum] (such as, GenBank Accession No.XP_(—)001950764 (SEQ ID NO:5)), phytoene dehydrogenase-like[Acyrthosiphon pisum] (such as, GenBank Accession No. XP_(—)001946689(SEQ ID NO:6)), and phytoene dehydrogenase-like [Acyrthosiphon pisum](such as, GenBank Accession No. XP_(—)001943938 (SEQ ID NO:7)).

Proteins involved in the synthesis and/or transport of biologicalpigments can be of any animal origin (such as mouse, porcine, human)including, but not limited to, melanocortin 1 receptor (such as, GenBankAccession No. EDL11741 (SEQ ID NO:8)), alpha melanocyte stimulatinghormones (MSH) (such as, SEQ ID NO:9), beta melanocyte stimulatinghormones (MSH) (such as, SEQ ID NO:10, SEQ ID NO:11), gamma melanocytestimulating hormones (MSH) (such as, SEQ ID NO:12), β-defensin (such as,GenBank Accession No. AAT67592 (SEQ ID NO:13)), agouti signaling proteinprecursor (such as, GenBank Accession No. NP_(—)056585 (SEQ ID NO:14)),tyrosinase (TYR) (such as, GenBank Accession No. BAA00341 (SEQ IDNO:15)), melanocyte-specific transporter protein (such as, GenBankAccession No. Q62052 (SEQ ID NO:16)), rab proteinsgeranylgeranyltransferase component A2 (such as, GenBank Accession No.NP_(—)067325 (SEQ ID NO:17)), ras-related protein Rab-7a (such as,GenBank Accession No. NP_(—)033031 (SEQ ID NO:18)), and probable E3ubiquitin-protein ligase (HERC2) (such as, GenBank Accession No.NP_(—)084390 (SEQ ID NO:19)).

In certain embodiments, proteins involved in the synthesis and/ortransport of biological pigments can be proteins having at least 80%identity, or having any percent identity higher than 80% (such as atleast 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%), to anyof SEQ ID NOs: 1-19.

Proteins Involved in the Texture, Structural Strength, and/or Length ofHair, Nail, Claw and/or Horn

In certain embodiment, the transgenic animal expresses a nucleic acidmolecule encoding a protein that alters hair quality (such as straightor curly hair) or length. In one embodiment, conditional over-expressionof WNT3 or DVL2 in the outer root sheath induces shorter hair inanimals. In certain embodiments, the transgenic animal expresses anucleic acid molecule encoding a protein involves that the texture,structural strength, and/or length of animal hair, nail, claw and/orhorn.

Proteins involved in controlling the texture, structural strength,and/or length of animal hair, nail, claw and/or horn include keratinproteins, including, but not limited to, keratin 1, keratin 2, keratin2A, keratin HB6, keratin 3, keratin 4, keratin 5, keratin 6, keratin 7,keratin 8, keratin 9, keratin 10, keratin 11, keratin 12, keratin 13,keratin 14, keratin 15, keratin 16, keratin 17, keratin 18, keratin 19,keratin 20, keratin 23, keratin 24, keratin 25, keratin 26, keratin 27,keratin 28, keratin 31, keratin 32, keratin 33, keratin 34, keratin 35,keratin 36, keratin 37, keratin 38, keratin 39, keratin 40, keratin 71,keratin 72, keratin 73, keratin 74, keratin 75, keratin 76, keratin 77,keratin 78, keratin 79, keratin 80, keratin 81, keratin 82, keratin 83,keratin 84, keratin 85, and keratin 86.

Modification of Amino Acid and/or Polynucleotide Sequences

The amino acid sequences of a variety of pigment proteins; proteinsinvolved in the synthesis and/or transport of pigments; proteinsinvolving the length and/or texture of animal skin or fur; and proteinsinvolved in the texture, structural strength, and/or length of animalnail, claw, and/or horn, are publically available, such as via theGenBank database. The present invention encompasses the use of suchproteins.

Polynucleotides and polypeptides within the scope of the subjectinvention can also be defined in terms of identity with those sequencesthat are specifically exemplified herein. The sequence identity willtypically be greater than 60%, preferably greater than 75%, morepreferably greater than 80%, even more preferably greater than 90%, andcan be greater than 95%. The identity of a sequence can be 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,or 99% as compared to a sequence exemplified herein.

Unless otherwise specified, as used herein percent sequence identity oftwo sequences can be determined using the algorithm of Karlin andAltschul (1990), modified as in Karlin and Altschul (1993). Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al. (1990). BLAST searches can be performed with the NBLASTprogram, score=100, wordlength=12, to obtain sequences with the desiredpercent sequence identity. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be used as described in Altschul et al.(1997). When utilizing BLAST and Gapped BLAST programs, the defaultparameters of the respective programs (NBLAST and XBLAST) can be used.See NCBI/NIH website.

Promoter Elements

In certain embodiments in accordance with the present invention, thenucleic acid molecule is operably linked to a constitutive, inducible,or tissue-specific promoter.

The term “constitutive promoter,” as used herein, refers to its ordinarymeaning that is an unregulated promoter that allows for continualtranscription of its associated gene. Constitutive promoters usefulaccording to the present invention include, but are not limited to,cytomegalovirus (CMV) promoter, CMV-chicken beta actin promoter,ubiquitin promoter, JeT promoter, SV40 promoter, beta globin promoter,elongation Factor 1 alpha (EF1-alpha) promoter, RSV promoter, andMo-MLV-LTR promoter.

Promoters useful according to the present invention include, but are notlimited to, universal promoters (e.g., Rosa26); tissue-specificpromoters, such as keratinocyte specific promoters (e.g., Keratin 14);melanocyte specific promoters (e.g., promoter of the melanocortin 1receptor (MCR1) gene); and dermal papilla-specific promoters. Promotersuseful according to the present invention include melanocyte specificpromoters and matrix-cell specific promoters.

Keratinocyte specific promoters include, but are not limited to,promoters of keratin 1, keratin 2, keratin 2A, keratin HB6, keratin 3,keratin 4, keratin 5, keratin 6, keratin 7, keratin 8, keratin 9,keratin 10, keratin 11, keratin 12, keratin 13, keratin 14, keratin 15,keratin 16, keratin 17, keratin 18, keratin 19, keratin 20, keratin 23,keratin 24, keratin 25, keratin 26, keratin 27, keratin 28, keratin 31,keratin 32, keratin 33, keratin 34, keratin 35, keratin 36, keratin 37,keratin 38, keratin 39, keratin 40, keratin 71, keratin 72, keratin 73,keratin 74, keratin 75, keratin 76, keratin 77, keratin 78, keratin 79,keratin 80, keratin 81, keratin 82, keratin 83, keratin 84, keratin 85,and keratin 86 gene.

In certain embodiments, promoters useful according to the presentinvention include, but are not limited to, promoters inducing geneexpression in the presence of an endogenous biological factor ofinterest, such as NF-KB, interferon-gamma, estrogen, and orglucocorticoids.

Promoters useful according to the present invention include, but are notlimited to, promoters inducing gene expression in the presence of aninfectious agent of interest, such as a virus, bacteria, and/orprotozoa.

In one embodiment, a den ial papilla-specific promoter is used forcreating customizable pigmentation, color, or pattern in cells derivedfrom somites; useful promoters include, but are not limited to, aRipply2 promoter, a Tabby promoter, and a Ticked promoter. The choice ofpromoters can be determined by performing multiple species comparisonsand/or using the extent of well-conserved promoter elements. In certainembodiments, the promoter element further comprises a nucleic acidmolecule encoding a reporter protein, which is expressed in response tothe administration of drugs, and/or a metabolic state or circulatinglevels of biomarkers in the transgenic animal. In one embodiment, thepromoter induces expression of a protein of interest (such as a pigmentprotein and/or a protein involved in the synthesis of a biologicalpigment) in response to the presence of a physiological state ofinterest in the transgenic animal, such as for example, cardiac stress,increased levels of circulating cytokines, and/or increased steroidpresence or activity.

Inducible Expression Systems

The inducible gene expression systems useful according the presentinvention include, but are not limited to, site-specific recombinationsystems including, but not limited to, a Cre-LoxP recombination system,a FLP-FRT recombination system; a tetracycline (Tet)-controlledtranscription activation system; an ecdysone inducible system; a heatshock on/off system; a lacO/IPTG system; a cumate repressor protein CymRsystem; a nitroreductase system; coumermycin/novobiocin-regulatedsystem; a RheoSwitch Ligand RSL1 system; a chimeric bipartite nuclearreceptor expression system; a GAL4 system; sterol or steroid orsynthetic steroid inducing/repressing system; and any combinationthereof.

In one embodiment, the inducible system useful according to the presentinvention is a Cre-LoxP recombination system. The genome of thetransgenic animal can comprise an exogenous nucleic acid molecule whoseexpression is under the control of a Cre/LoxP recombination system,wherein the Cre/LoxP recombination system prevents the expression of theexogenous nucleic acid molecule. In one specific embodiment, theCre/LoxP recombination system comprises a lox-stop-lox (LSL) sequence.

The Cre-LoxP recombination system is a site-specific recombinationtechnology useful for performing site-specific deletions, insertions,translocations, and inversions in the DNA of cells or transgenicanimals. The Cre recombinase protein (encoded by the locus originallynamed as “causes recombination”) consists of four subunits and twodomains: a larger carboxyl (C-terminal) domain and a smaller amino(N-terminal) domain. The loxP (locus of X-over P1) is a site on theBacteriophage P1 and consists of 34 bp. The results ofCre-recombinase-mediated recombination depend on the location andorientation of the loxP sites, which can be located cis or trans. Incase of cis-localization, the orientation of the loxP sites can be thesame or opposite. In case of trans-localization, the DNA strandsinvolved can be linear or circular. The results of Crerecombinase-mediated recombination can be excision (when the loxP sitesare in the same orientation) or inversion (when the loxP sites are inthe opposite orientation) of an intervening sequence in case of cis loxPsites, or insertion of one DNA into another or translocation between twomolecules (chromosomes) in case of trans loxP sites. The Cre-LoxPrecombination system is known in the art, see, for example, Andras Nagy,Cre recombinase: the universal reagent for genome tailoring, Genesis26:99-109 (2000).

The Lox-Stop-T ox (LSL) cassette prevents expression of the transgene inthe absence of Cre-mediated recombination. In the presence of Crerecombinase, the LoxP sites recombine, and the stop cassette is deleted.The Lox-Stop-Lox (LSL) cassette is known in the art. See, AllenInstitute for Brain Science, Mouse Brain Connectivity Altas, TechnicalWhite Paper: Transgenic Characterization Overview (2012).

In certain embodiments, the loxP site further comprises a reporter geneencoding gene (e.g., lacz, GFP) and/or a nucleic acid molecule encodinga second pigmentation protein (e.g., if the first pigmentation containsa dominantly active MC1R, another agouti gene could be flanked by theloxP site).

Tetracycline (Tet)-controlled transcriptional activation is a method ofinducible expression where transcription is reversibly controlled by thepresence or absence of the antibiotic tetracycline or one of itsderivatives (e.g., doxycycline). Gene expression is activated as aresult of binding of the Tet-off or Tet-on protein to tetracyclineresponse elements (TREs) located within an inducible promoter. Both theTet-on and Tet-off proteins activate gene expression. The Tet-Offprotein activates gene expression in the absence of a tetracyclinederivative-doxycycline (Dox), whereas the Tet-on protein activates geneexpression in the presence of Dox.

In the Tet-off system, the tetracycline transactivator (tTA) protein,which is created by fusing the TetR (tetracycline repressor) protein(obtainable from Escherichia coli bacteria) with the VP16 protein(obtainable from the Herpes Simplex Virus), binds on DNA at a TetOoperator. Once bound the TetO operator activates the promoter coupled tothe TetO operator, thereby activating the transcription of the nearbygene. Tetracycline derivatives bind tTA and render it incapable ofbinding to TRE sequences, thereby preventing transactivation of targetgenes.

In the Tet-On system, when the tTA protein is bound by doxycycline, thedoxycycline-bound tTA is capable of binding the TetO operator. Thus, theintroduction of doxycyline to the system initiates the transcription ofthe genetic product. The Tet-on system is sometimes preferred for thefaster responsiveness.

The reverse tTA (rtTA) is a complementary genetic module for rapid geneactivation by addition of Dox (Tet-on). See Kistner et al.,Doxycycline-mediated quantitative and tissue-specific control of geneexpression in transgenic mice, Proc. Natl. Acad. Sci. U.S.A. Vol. 93,pp. 10933-10938 (1996).

The Tet-on advanced transactivator (also known as rtTA2^(s)-M2) is analternative version of Tet-On that shows reduced basal expression, andfunctions at a 10-fold lower Dox concentration than Tet-on. In addition,its expression is considered to be more stable in eukaryotic cells dueto being human codon optimized and utilizing three minimaltranscriptional activation domains. Tet-on 3G (also known as rtTA-V10)is similar to Tet-on Advanced, and is human codon optimized and composedof three minimal VP 16 activation domains. The Tet-on 3G is sensitive to100-fold less Dox than the original Tet-on.

In one embodiment of a tetracycline-responsive regulatory expressionelement, a tetracycline-controlled reverse transactivator (rtTA)comprises a tetR (e.g., from Escherichia coli Tn10); a mammaliantranscription factor VP 16 transactivating domain serving as aneffector; and a tissue-specific promoter controlling the rtTA effectortranscription. In the presence of doxycycline, the rtTA binds to a(TeTO)₇ operator (a seven tandemly repeated TetO sequence) placedupstream of a CMV promoter that drives expression of a transgene. As aresult, transgene expression can be switched on or off by administrationand withdrawal of doxycycline.

Expression Constructs

The present invention also provides expression constructs, vectors, aswell as host cells useful for producing transgenic animals.

As used herein, the term “expression construct” refers to a combinationof nucleic acid sequences that provides for transcription of an operablylinked nucleic acid sequence. Expression constructs of the inventionalso generally include regulatory elements that are functional in theintended host cell in which the expression construct is to be expressed.Thus, a person of ordinary skill in the art can select regulatoryelements for use in, for example, bacterial host cells, yeast hostcells, plant host cells, insect host cells, mammalian host cells, andhuman host cells. Regulatory elements include promoters, transcriptiontermination sequences, translation termination sequences, enhancers, andpolyadenylation elements.

In certain embodiments, the present invention provides expressionconstructs for customizing color and/or pattern of animals, includingpigmentation constructs and patterning constructs.

FIG. 1 shows an embodiment of a pigmentation construct comprising apromoter, a loxP cassette, a nucleic acid molecule encoding a pigmentprotein of interest or a protein involved in the synthesis and/ortransport of a pigment of interest, and polyadenylation sequence. In oneembodiment of the pigmentation construct, the expression of the pigmentprotein of interest or a protein involved in the synthesis and/ortransport of a pigment of interest is activated by application of Crerecombinase.

In another embodiment, the present invention provides a patternconstruct. FIG. 2 shows an embodiment of a patterning construct. In oneembodiment, the promoter of the patterning construct is derived from agene specific to somite boundary specification. In one embodiment, thepromoter is selected from a Ripply2 promoter, a Tabby promoter, and aTicked promoter. In one embodiment, the transgenic animal comprises apigmentation construct and a patterning construct. In one embodiment,the transgenic animal has customizable constitutive vertical stripes onthe dorsal dermis.

In one embodiment, the genome of the transgenic animal comprises:

1) pigmentation construct as shown in FIG. 1, wherein the promoter is amelanocyte-specific promoter and the nucleic acid molecule encodes aprotein involved in the synthesis of a red pigment (such as ASIP orMC1R);

2) a pigmentation construct as shown in FIG. 1, wherein the promoter isa dermal papilla-specific promoter and the nucleic acid moleculeencodes—defensin 103; and

3) a set of constructs as shown in FIG. 3, wherein the promoter is amelanocyte-specific promoter.

An expression construct of the invention can comprise a promotersequence operably linked to a polynucleotide sequence encoding a peptideof the invention. Promoters can be incorporated into a polynucleotideusing standard techniques known in the art. Multiple copies of promotersor multiple promoters can be used in an expression construct of theinvention. In a preferred embodiment, a promoter can be positioned aboutthe same distance from the transcription start site as it is from thetranscription start site in its natural genetic environment. Somevariation in this distance is permitted without substantial decrease inpromoter activity. A transcription start site is typically included inthe expression construct.

As used herein, the term “operably linked” refers to a juxtaposition ofthe components described wherein the components are in a relationshipthat permits them to function in their intended manner. In general,operably linked components are in contiguous relation. Sequence(s)operably-linked to a coding sequence may be capable of effecting thereplication, transcription and/or translation of the coding sequence.For example, a coding sequence is operably-linked to a promoter when thepromoter is capable of directing transcription of that coding sequence.

A “coding sequence” or “coding region” is a polynucleotide sequence thatis transcribed into mRNA and/or translated into a polypeptide. Forexample, a coding sequence may encode a polypeptide of interest. Theboundaries of the coding sequence are determined by a translation startcodon at the 5′-terminus and a translation stop codon at the3′-terminus.

The term “promoter,” as used herein, refers to a DNA sequence operablylinked to a nucleic acid sequence to be transcribed such as a nucleicacid sequence encoding a desired molecule. A promoter is generallypositioned upstream of a nucleic acid sequence to be transcribed andprovides a site for specific binding by RNA polymerase and othertranscription factors. In specific embodiments, a promoter is generallypositioned upstream of the nucleic acid sequence transcribed to producethe desired molecule, and provides a site for specific binding by RNApolymerase and other transcription factors.

In addition to a promoter, one or more enhancer sequences may beincluded such as, but not limited to, cytomegalovirus (CMV) earlyenhancer element and an SV40 enhancer element. Additional includedsequences are an intron sequence such as the beta globin intron or ageneric intron, a transcription termination sequence, and an mRNApolyadenylation (pA) sequence such as, but not limited to, SV40-pA,beta-globin-pA, the human growth hormone (hGH) pA and SCF-pA.

In one embodiment, the expression construct comprises polyadenylation sequences, such as polyadenylation sequences derived from bovine growthhormone (BGH) and SV40.

The term “polyA” or “p(A)” or “pA” refers to nucleic acid sequences thatsignal for transcription termination and mRNA polyadenylation. The polyAsequence is characterized by the hexanucleotide motif AAUAAA. Commonlyused polyadenylation signals are the SV40 pA, the human growth hormone(hGH) pA, the beta-actin pA, and beta-globin pA. The sequences can rangein length from 32 to 450 bp. Multiple pA signals may be used.

In one embodiment, the genetic construct comprises a nucleic acidmolecule encoding a selection marker, such as neomycin resistancebiomarker protein, which can be excised through PIGGYBAC™ transposons.In one embodiment, the construct is flanked by short homology arms.

The term “vector” is used to refer to any molecule (e.g., nucleic acid,plasmid, or virus) used to transfer coding information (e.g., apolynucleotide of the invention) to a host cell.

The terms “expression vector” and “transcription vector” are usedinterchangeably to refer to a vector that is suitable for use in a hostcell (e.g., a subject's cell) and contains nucleic acid sequences thatdirect and/or control the expression of exogenous nucleic acidsequences. Expression includes, but is not limited to, processes such astranscription, translation, and RNA splicing, if introns are present.Vectors useful according to the present invention include plasmids,viruses, BACs, YACs, and the like. Particular viral vectorsillustratively include those derived from adenovirus, adeno-associatedvirus and lentivirus.

As used herein, the term “isolated” molecule (e.g., isolated nucleicacid molecule) refers to molecules which are substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized.

The term “recombinant” is used to indicate a nucleic acid construct inwhich two or more nucleic acids are linked and which are not foundlinked in nature.

The term “nucleic acid” as used herein refers to RNA or DNA moleculeshaving more than one nucleotide in any form including single-stranded,double-stranded, oligonucleotide or polynucleotide.

The term “nucleotide sequence” is used to refer to the ordering ofnucleotides in an oligonucleotide or polynucleotide in a single-strandedform of nucleic acid.

The term “expressed” refers to transcription of a nucleic acid sequenceto produce a corresponding mRNA and/or translation of the mRNA toproduce the corresponding protein. Expression constructs can begenerated recombinantly or synthetically or by DNA synthesis usingwell-known methodology.

The term “regulatory element” as used herein refers to a nucleotidesequence which controls some aspect of the expression of an operablynucleic acid sequence Exemplary plry regulatory elements illustrativelyinclude an enhancer, an internal ribosome entry site (IRES), an intron,an origin of replication, a polyadenylation signal (pA), a promoter, atranscription termination sequence, and an upstream regulatory domain,which contribute to the replication, transcription, post-transcriptionalprocessing of a nucleic acid sequence. Those of ordinary skill in theart are capable of selecting and using these and other regulatoryelements in an expression construct with no more than routineexperimentation.

In one embodiment, the construct of the present invention comprises aninternal ribosome entry site (IRES). In one embodiment, the expressionconstruct comprises kozak consensus sequences.

Optionally, a reporter gene is included in the transgene construct. Theterm “reporter gene” as used herein refers to a gene that is easilydetectable when expressed, for example, via chemiluminescence,fluorescence, colorimetric reactions, antibody binding, induciblemarkers, ligand binding assays, and the like. Exemplary reporter genesinclude but are not limited to green fluorescent protein. The productionof recombinant nucleic acids, vectors, transformed host cells, proteinsand protein fragments by genetic engineering is well known.

If desired, the vector may optionally contain flanking nucleic sequencesthat direct site-specific homologous recombination. The use of flankingDNA sequences to permit homologous recombination into a desired geneticlocus is known in the art. At present it is preferred that up to severalkilobases or more of flanking DNA corresponding to the chromosomalinsertion site be present in the vector on both sides of the encodingsequence (or any other sequence of this invention to be inserted into achromosomal location by homologous recombination) to assure precisereplacement of chromosomal sequences with the exogenous DNA. See e.g.Deng et al, 1993, Mol. Cell. Biol 13(4):2134-40; Deng et al, 1992, MolCell Biol 12(8):3365-71; and Thomas et al, 1992, Mol Cell Biol12(7):2919-23. It should also be noted that the cell of this inventionmay contain multiple copies of the gene of interest.

Transformed host cells are cells which have been transformed ortransfected with vectors containing nucleic acid constructs of theinvention and may or may not transcribe or translate the operativelyassociated nucleic acid of interest.

RNA Interference Cassette for Customization of Animal Traits

In another embodiment, the present invention provides a transgenicanimal with customizable traits, wherein the genome of the transgenicanimal comprises:

an exogenous inhibitory RNA coding sequence of interest, operably linkedto a promoter and under the control of an inducible gene expressionsystem that requires the presence of an inducing agent to activate geneexpression;

wherein the expression of the exogenous inhibitory RNA coding sequenceof interest is inhibited in the absence of the inducing agent.

In certain embodiments, the exogenous inhibitory RNA coding sequence ofinterest interferes with the expression of a nucleic acid sequenceencoding pigment proteins; proteins involved in the synthesis and/ortransport of pigments; proteins involving the length and/or texture ofanimal skin or fur; luminescent (such as fluorescent) proteins; andproteins involved in the texture, structural strength, and/or length ofanimal nail, claw, or horn texture.

In one embodiment, an exogenous inhibitory RNA coding sequence encodesan siRNA that interferes with the expression of cross-linking actin) inthe nails, thereby producing genetically-engineered animals (such ascats) with nails that are soft instead of sharp.

In one embodiment, the RNAi construct comprises an siRNA that interfereswith the expression of a nucleic acid molecule encoding cross-linkingkeratin), operably linked to a promoter specific to the cross-linkingkeratin, and is under the control of a reporter gene flanked by loxPsites.

Keratin proteins involved in the texture, structural strength, and/orlength of animal hair, nail, claw and/or horn include, but are notlimited to, keratin 1, keratin 2, keratin 2A, keratin HB6, keratin 3,keratin 4, keratin 5, keratin 6, keratin 7, keratin 8, keratin 9,keratin 10, keratin 11, keratin 12, keratin 13, keratin 14, keratin 15,keratin 16, keratin 17, keratin 18, keratin 19, keratin 20, keratin 23,keratin 24, keratin 25, keratin 26, keratin 27, keratin 28, keratin 31,keratin 32, keratin 33, keratin 34, keratin 35, keratin 36, keratin 37,keratin 38, keratin 39, keratin 40, keratin 71, keratin 72, keratin 73,keratin 74, keratin 75, keratin 76, keratin 77, keratin 78, keratin 79,keratin 80, keratin 81, keratin 82, keratin 83, keratin 84, keratin 85,and keratin 86.

In one embodiment, the present invention provides a microRNA cassettecomprising the siRNA coding sequence and a 3′ UTR sequence.

As used herein, the term “RNA interference” (“RNAi”) refers to aselective intracellular degradation of RNA. RNAi occurs in cellsnaturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAiproceeds via fragments cleaved from free dsRNA which direct thedegradative mechanism to other similar RNA sequences. Alternatively,RNAi can be initiated by the hand of man, for example, to silence theexpression of endogenous target genes, such as PKC-t.

As used herein, the term “small interfering RNA” (“siRNA”) (alsoreferred to in the art as “short interfering RNAs”) refers to an RNA (orRNA analog) comprising between about 10-50 nucleotides (or nucleotideanalogs) which is capable of directing or mediating RNA interference.

As used herein, a siRNA having a “sequence sufficiently complementary toa target mRNA sequence to direct target-specific RNA interference(RNAi)” means that the siRNA has a sequence sufficient to trigger thedestruction of the target mRNA (e.g., PKC-i mRNA) by the RNAi machineryor process. “mRNA” or “messenger RNA” or “transcript” is single-strandedRNA that specifies the amino acid sequence of one or more polypeptides.This information is translated during protein synthesis when ribosomesbind to the mRNA.

The term “nucleotide” refers to a nucleoside having one or morephosphate groups joined in ester linkages to the sugar moiety. Exemplarynucleotides include nucleoside monophosphates, diphosphates andtriphosphates. The terms “polynucleotide” and “nucleic acid molecule”are used interchangeably herein and refer to a polymer of nucleotidesjoined together by a phosphodiester linkage between 5′ and 3′ carbonatoms. The terms “nucleic acid” or “nucleic acid sequence” encompass anoligonucleotide, nucleotide, polynucleotide, or a fragment of any ofthese, DNA or RNA of genomic or synthetic origin, which may besingle-stranded or double-stranded and may represent a sense orantisense strand, peptide nucleic acid (PNA), or any DNA-like orRNA-like material, natural or synthetic in origin. As will be understoodby those of skill in the art, when the nucleic acid is RNA, thedeoxynucleotides A, G, C, and T are replaced by ribonucleotides A, G, C,and U, respectively.

As used herein, the term “RNA” or “RNA molecule” or “ribonucleic acidmolecule” refers generally to a polymer of ribonucleotides. The term“DNA” or “DNA molecule” or deoxyribonucleic acid molecule” refersgenerally to a polymer of deoxyribonucleotides. DNA RNA molecules canmolecules can be synthesized naturally (e.g., by DNA replication ortranscription of DNA, respectively). RNA molecules can bepost-transcriptionally modified. DNA and RNA molecules can also bechemically synthesized. DNA and RNA molecules can be single-stranded(i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g., doublestranded, i.e., dsRNA and dsDNA, respectively). Based on the nature ofthe invention, however, the term “RNA” or “RNA molecule” or “ribonucleicacid molecule” can also refer to a polymer comprising primarily (i.e.,greater than 80% or, preferably greater than 90%) ribonucleotides butoptionally including at least one non-ribonucleotide molecule, forexample, at least one deoxyribonucleotide and/or at least one nucleotideanalog.

As used herein, the term “nucleotide analog”, also referred to herein asan “altered nucleotide” or “modified nucleotide,” refers to anon-standard nucleotide, including non-naturally occurringribonucleotides or deoxyribonucleotides. Preferred nucleotide analogsare modified at any position so as to alter certain chemical propertiesof the nucleotide yet retain the ability of the nucleotide analog toperform its intended function.

As used herein, the term “RNA analog” refers to a polynucleotide (e.g.,a chemically synthesized polynucleotide) having at least one altered ormodified nucleotide as compared to a corresponding unaltered orunmodified RNA but retaining the same or similar nature or function asthe corresponding unaltered or unmodified RNA. As discussed above, theoligonucleotides may be linked with linkages which result in a lowerrate of hydrolysis of the RNA analog as compared to an RNA molecule withphosphodiester linkages. Exemplary RNA analogues include sugar- and/orbackbone-modified ribonucleotides and/or deoxyribonucleotides. Suchalterations or modifications can further include addition ofnon-nucleotide material, such as to the end(s) of the RNA or internally(at one or more nucleotides of the RNA). An RNA analog need only besufficiently similar to natural RNA that it has the ability to mediate(mediates) RNA interference or otherwise reduce target gene expression.

Methods of Making Transgenic Non-Human Animals

Any of various methods can be used to introduce a transgene into anon-human animal to produce a transgenic animal. Such techniques arewell-known in the art and include, but are not limited to, pronuclearmicroinjection, viral infection and transformation of embryonic stemcells and iPS cells. Methods for generating transgenic animals that canbe used include, but are not limited to, those described in J. P.Sundberg and T. Ichiki, Eds., Genetically Engineered Mice Handbook, CRCPress; 2006; M. H. Hofker and I. van Deursen, Eds., Transgenic MouseMethods and Protocols, Humana Press, 2002; A. L. Joyner, Gene Targeting:A Practical Approach, Oxford University Press, 2000; Manipulating theMouse Embryo: A Laboratory Manual, 3rd edition, Cold Spring HarborLaboratory Press; 2002, ISBN-10: 0879695919; K. Turksen (Ed.), Embryonicstem cells: methods and protocols in Methods Mol. Biol. 2002; 185,Humana Press; Current Protocols in Stem Cell Biology, ISBN:978047015180; Meyer et al. PNAS USA, vol. 107 (34), 15022-15026.

In certain embodiments, the genetically engineered animals withsite-specific knock-ins can be created using spermatogonial stem cells(SSCs), piggyBac™ mobile DNA technology using transposable elements,Xanthamonas transcription activator-like (TAL) Nucleases (XTNs) [akaTAL-effector nucleases (TALENs)], and a combination thereof.

Methods for Customizing Animal Traits

In one embodiment, the present invention provides a method ofcustomizing animal traits using the transgenic animal of the invention.In one embodiment, the method comprises:

a) providing a transgenic animal whose genome comprises:

an exogenous nucleic acid molecule encoding a protein of interest,wherein the nucleic acid molecule is operably linked to a promoter andis under the control of an inducible gene expression system thatrequires the presence of an inducing agent to activate gene expression;

wherein the expression of the exogenous nucleic acid molecule isinhibited in the absence of the inducing agent;

b) administering the inducing agent to the transgenic animal therebyinducing the expression of the exogenous nucleic acid molecule.

In certain embodiments, the exogenous nucleic acid molecule encodes aprotein of interest, wherein the protein of interest is selected frompigment proteins; proteins involved in the synthesis and/or transport ofpigments; luminescent (such as fluorescent) proteins; proteins involvingthe length and/or texture of animal skin or fur; and proteins involvedin the texture, structural strength, and/or length of animal nail, claw,and/or horn.

In certain embodiments, the inducible gene expression systems usefulaccording the present invention include, but are not limited to,site-specific recombination systems including, but not limited to, aCre-LoxP recombination system, a FLP-FRT recombination system; atetracycline (Tet)-controlled transcription activation system; anecdysone inducible system; a heat shock on/off system; a lacO/IPTGsystem; a cumate repressor protein CymR system; a nitroreductase system;coumermycin/novobiocin-regulated system; a RheoSwitch Ligand RSL1system; a chimeric bipartite nuclear receptor expression system; a GAL4system; sterol or steroid or synthetic steroid inducing/repressingsystem; and any combinations thereof.

In certain embodiments, the inducing agents for gene expression usefulaccording the present invention include, but are not limited to, crerecombinase, HTCre; FLP recombinase; tetracycline or its derivativessuch as doxycycline; ecdysone; cumate; nitroreductase steroids; and anycombinations thereof.

In one embodiment, the transgenic animal comprises an exogenous nucleicacid molecule that is under the control of a site-specific recombinationsystem, and the expression of the exogenous nucleic acid molecule isinduced after the administration (such as via topical administration) ofa recombinase protein, or the administration (such as via injection) ofa nucleic molecule encoding a recombinase protein, to the transgenicanimal.

In one embodiment, the genome of the transgenic animal comprises anexogenous nucleic acid molecule whose expression is under the control ofa Cre/LoxP recombination system, wherein the Cre/LoxP recombinationsystem prevents the expression of the exogenous nucleic acid molecule,wherein the administration (such as via topical administration) of Crerecombinase and/or HTCre, or the administration (such as via injection)of a nucleic molecule encoding Cre recombinase and/or HTCre, to thetransgenic animal, induces the expression of the exogenous nucleic acidmolecule, thereby customizing the animal trait(s) of interest.

In another specific embodiment, the method of customizing animal traitscomprises:

a) providing a transgenic animal whose genome comprises:

a first exogenous nucleic acid molecule encoding a protein of interest(such as pigmentation protein) operably linked to a first promoter andunder the control of a loxP site, wherein the loxP site prevents theexpression of the first nucleic acid molecule in the absence of Crerecombinase protein;

a second nucleic acid encoding a reverse tTA (rtTA), operably linked toa second promoter; and

a third nucleic acid molecule encoding a Cre recombinase protein,operably linked to a third promoter under the control of a TetOoperator;

b) administering doxycycline to the transgenic animal, thereby inducingthe expression of the exogenous nucleic acid molecule.

The inducing agent for administration to the transgenic animal can be ina foam that can be combined with a carrier. The term “carrier” refers toa diluent, adjuvant, excipient, or vehicle with which the compound isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum oil such as mineral oil,vegetable oil such as peanut oil, soybean oil, and sesame oil, animaloil, or oil of synthetic origin. Saline solutions and aqueous dextroseand glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions.

The inducing agent and compositions can be administered to thetransgenic animal by standard routes, including oral, inhalation, orparenteral administration including intravenous, subcutaneous, topical,transdermal, intradermal, transmucosal, intraperitoneal, intramuscular,intracapsular, intraorbital, intracardiac, transtracheal, subcutaneous,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal,epidural and intrasternal injection, infusion, and electroporation, aswell as co-administration as a component of any medical device or objectto be inserted (temporarily or permanently) into a transgenic animal.

EXAMPLES

Following are examples that illustrate procedures and embodiments forpracticing the invention. The examples should not be construed aslimiting.

Example 1 Customization of Skin or Fur Pigmentation in Animals

This Example provides embodiments of genetic constructs for customizingskin and fur pigmentation in animals.

Animals with Customized Pigmentation and Pattern in the Skin or Fur

C3H/HeJ murine strain with a brown (“Agouti” coloration) skin color aregenetically engineered to express the following three constructs: 1) aconstruct comprising a keratin-14 specific promoter, a loxp cassettecomprising a nucleic acid encoding a red fluorescent protein, a pigmentcassette comprising a nucleic acid encoding a dominant black (ΔG23) betadefensin 103 protein, and an SV40 (with intron) polyadenylationsequence; 2) a construct comprising a nucleic acid molecule encoding areverse tetracycline transactivator (rtTA), operably linked to akeratin-14 specific promoter that initiates the transcription of thenucleic acid encoding rtTA, and an SV40 polyadenylation sequence; and 3)a construct comprising an agouti signaling protein (ASP), operablylinked to a tetracycline-sensitive promoter (TetO)7 that initiates thetranscription of the nucleic acid molecule encoding ASP, and a bovinegrowth hormone (BGH) polyadenylation sequence.

When doxycycline is fed to the genetically-modified mice, mouse furturns golden (this is the first demonstration of genetic modification ofhair color after birth). The application of Cre or HTNCre to the shavedskin of the genetically-engineered mice, via a carrier base (e.g.,protein carriers, such as lipid bilayers), induces genetically permanentblack coloration of fur in the area where Cre and/or HTNCre is applied.In accordance with the present invention, mice born with brown fur canbe modified to have golden fur with arbitrarily shaped black markings.For example, a black name or black logo can be created on mice fur witha gold background.

To customize skin pigmentation in animals, the animal could only expressa pigmentation construct, and need not express a patterning construct(as shown in FIG. 2). The customization of pigmentations and patterns isactivated directly by application of Cre and/or HTNCre.

In one embodiment heritable patterns can be created by geneticallymodifying the animals to express a patterning construct. FIG. 2 showsone embodiment of a patterning construct. The promoter can be theRipply2 promoter, or from any gene specific to somite boundaryspecification. Alternate promoters could be the Tabby or Tickedpromoters.

In one embodiment, the genetically-modified animal, whose genomecomprises a pigmentation construct and a patterning construct, hasconstitutive vertical stripes on the dorsal dermis.

Complex/Multicolored Patterns

FIG. 3 illustrates certain embodiments of genetic constructs forcreating complex or multicolored patterns in animal skin or fur. In oneembodiment, complex patterns can be created with the use of a constructcomprising an inducible system, such as promoters with mechanisms ofinducibility. As shown in FIG. 3, Cre is activated by the presence ofdoxycycline only in tissues specific for the rtTA promoter. The use oftissue-specific promoter maintains somite border in animals; forexample, the transgene can only be activated during the developmentalperiod. Inducible system can also be used to create multiple colors.

Inducible systems useful according to the present invention include, butare not limited to, tetracycline, ecdysone, and tamoxifen induciblesystems; FLP-FRT recombination system; and Cre-LOX recombination system.

Example 2 Mice with Customized Fur Color and Pattern

This Example shows the creation of genetically-modified mice whose furcolor can be permanently altered through the transdermal application ofHTNCre—a recombinase that can easily cross cell membranes.

FIG. 4 shows a genetic construct for creating customized patterns andcolor in mouse skin or fur. The construct comprises a Keratinl4promoter, which is expressed in all skin fibroblasts, to drive thedominant black form of signaling molecule beta-Defl 03; the expressionof beta-Def103 is blocked by a Loxp excisable nucleic acid encoding ringfinger protein (RFP) (the RFP is used as a marker).

Agouti mice are genetically-engineered to express a transgene encodingthe “dominant black” signaling molecule βDef103, and the transgeneexpression is activated by the application of recombinase.

FIGS. 5A and B are photographs that show two genetically-engineered micein which the expression of the dominant black pigment protein isactivated by dermal or intradermal application of HTNCre in a carriersolution. Before the present invention, recombinase has never beenapplied in live animals.

FIG. 6 shows that the application of recombinase togenetically-engineered mice can result in LW 3-dependent change in furcolor. The Agouti hairs are normally characterized by cells comprisingyellow pheomelanin (A). As shown in FIG. 6, through increasingactivation of the transgene by increasing recombinase doses, the mousefur pigments can be shifted to a mix of pheomelanin and black eumelanin(B) to pure eumelanin (C) to so much melanin that the compartmentalizedstructure breaks down (D). This Example shows that transdermalapplication of recombinase can result in fine control of hair color ingenetically-engineered mice.

Example 3 Customized Skin Color and Pattern for Cattle Identification

In the cattle industry, a robust method of birth processing forindividual identification has become increasingly important for proof ofownership, herd management, tracking of animal movements, and perhaps,most importantly, animal disease traceability.

This Example provides transgenic cattle having customized skin color andpatterns that can be used as a code (e.g., bar code) for cattleidentification. The transgenic cattle can be created using the methoddescribed in Example 2. As shown in Example 2, transdermal orintradermal application of recombinase to transgenic mice whose genomiccomprises a Cre-LoxP recombination system induce customizable changes incoat color after birth.

FIG. 7 shows a construct design for creating customized pattern or coloridentification in cattle. The construct comprises a nucleic acidmolecule encoding a dominant negative Rab7, operably linked to a MC1Rpromoter and under the control of the loxP-STOP-loxP sequence. Theexpression of the Rab7 can be induced by the application of Crerecombinase. The use of TAL nucleases allows site-specific knock-inswith short homology arms. The acrosin promoter/EGFP arm on the constructallows flow sorting for genetically modified sperm, thereby ensuring100% genetically modified offspring in the F1 generation. An importantelement for the construct is the MC1R (melanocortin receptor) promoter,which drives the expression of a dominant negative Rab7 gene only whenactivated with recombinase. The combination of various elements in theconstruct allows creation of cattle modified with site-specificknock-in(s) that allows creation of permanent identification informationon cattle.

FIG. 8 shows a depiction of a Black Angus heifer genetically engineeredto express a customizable identification pattern in the skin.

During birth processing, individual identification information can beeasily created by applying Cre recombinase to cattle whose genomecomprises the construct shown in FIG. 7. Also, the transgenes are onlyexpressed in the animal skin, which can be removed during foodprocessing; therefore, the creation of cattle identification in theanimal skin should not raise regulatory issues with governmentalagencies such as USDA.

In certain embodiments, site-specific knock-in animals can be createdusing conventional technologies, including, but not limited to,spermatogonial stem cells (SSCs), piggyBac™ mobile DNA technology usingtransposable elements, Xanthamonas transcription activator-like (TAL)Nucleases (XTNs) [aka TAL-effector nucleases (TALENs)], and acombination thereof.

In one embodiment, Black Angus coat color is post-natally modified witha heterozygous knock-in using a melanocyte-specific dominant negativeRab7, which is required for intracellular transport of the criticalmelanogenesis gene Tyrp1.

Example 4 Customized Skin Color and Pattern for Cattle Disease Detection

Diseases are a concern for nearly every beef and dairy producer, andmany common diseases can dramatically impact production without havingovert clinical signs. Sudden death is often the first and only sign ofclostridial diseases. Subclinical mastitis is a common and expensiveproblem in dairy production. Even rumors of bovine spongioformencephalopathy (BSE), which rarely has overt clinical signs, can causesevere economic damage to beef industry. In addition to infectiousdiseases, metabolic diseases in cattle can also cause economic damages:⅓ of beef cattle have subclinical copper deficiency due to the presenceof chelating agents in their diet. Changes in fur or skin color, inaccordance with the present invention (e.g., using recombinase-activatedcoat-color specific markers) can be used to identify the presence ofdiseases in cattle.

In one embodiment, the construct for cattle disease detection isidentical to the construct as shown in FIG. 7, except that the MC1Rpromoter is replaced with an alternate recombinase system (e.g., Flp-Frtrecombination system) and/or disease-specific promoters, such aspromoters responsive to NFkB, Ifnγ, or copper-deficiency

In one embodiment, during birth processing, cattle are painted with therecombinase to create the desired symbol. The painted area would changecolor once the cattle develop inflammatory or metabolic stresses (theexpression of pigment protein regulated by disease-specific promoter).The cattle disease detection application can be used in combination withthe cattle identification application through the use of differentrecombinases and recombinase targets.

Example 5 Dogs with Customizable Coats

Different dog breeds have coat color as a result of differentcombinations of mutations in pigment genes. Pugs have an intactmelanocortin receptor with no endogenous expression of the “dominantblack” signaling molecule βDef103.

Dogs with customized fur color and pattern can be created using themethods described in Examples 3 and 4 for cattle, or through the processshown in Example 2 for mice. In one embodiment, genetically-engineeredpugs whose genome comprises the construct as shown in FIG. 7 have furwith customizable color and pattern including tiger stripes, logos,writing, and hearts.

Dogs are normally difficult to genetically engineer due to the odditiesin the egg development system in canines. Currently,genetically-engineered dogs cannot be created by in vitro fertilization(IVF). Genetically-engineered dogs with site-specific knock-ins can becreated using spennatogonial stem cells (SSCs), piggyBac™ mobile DNAtechnology using transposable elements, Xanthamonas transcriptionactivator-like (TAL) Nucleases (XTNs) [aka TAL-effector nucleases(TALENs)], and a combination thereof.

All references, including publications, patent applications and patents,cited herein are hereby incorporated by reference to the same extent asif each reference was individually and specifically indicated to beincorporated by reference and was set forth in its entirety herein.

The terms “a” and “an” and “the” and similar referents as used in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. Unless otherwise stated, all exact valuesprovided herein are representative of corresponding approximate values(e.g., all exact exemplary values provided with respect to a particularfactor or measurement can be considered to also provide a correspondingapproximate measurement, modified by “about,” where appropriate).

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise indicated. No language in the specification should beconstrued as indicating any element is essential to the practice of theinvention unless as much is explicitly stated.

The description herein of any aspect or embodiment of the inventionusing terms such as “comprising”, “having”, “including” or “containing”with reference to an element or elements is intended to provide supportfor a similar aspect or embodiment of the invention that “consists of”,“consists essentially of”, or “substantially comprises” that particularelement or elements, unless otherwise stated or clearly contradicted bycontext (e.g., a composition described herein as comprising a particularelement should be understood as also describing a composition consistingof that element, unless otherwise stated or clearly contradicted bycontext).

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

REFERENCES

-   1. Candille S I, Kaelin C B, Cattanach B M, Yu B, Thompson D A, Nix    M A, Kerns J A, Schmutz S M, Millhauser G L, and Barsh G S. A    -defensin mutation causes black coat color in domestic dogs. Science    318: 1418-1423, 2007.-   2. Kistner et al., Dixycycline-mediated quantitative and    tissue-specific control of gene expression in transgenic mice, Proc.    Natl. Acad. Sci. U.S.A. Vol. 93, pp. 10933-10938, 1996.-   3. Nolden L, Edenhofer F, Haupt S, Koch P, Wunderlich FT, Siemen H,    and Brustle O. Site-specific recombination in human embryonic stem    cells induced by cell-permeant Cre recombinase. Nat Methods 3:    461-467, 2006.-   4. Nagy, Cre recombinase: the universal reagent for genome    tailoring, Genesis 26:99-109, 2000.

I claim:
 1. A cell of a transgenic non-human animal, wherein the cellcomprises: an exogenous nucleic acid molecule operably linked to apromoter, wherein the expression of the exogenous nucleic acid moleculeis under the control of an inducible expression system that requires thepresence of an inducing agent to activate expression; and wherein theexpression of the exogenous nucleic acid molecule is inhibited in theabsence of the inducing agent; wherein the exogenous nucleic acidmolecule is selected from: a nucleic acid molecule encoding a proteinselected from pigment proteins; proteins involved in the synthesisand/or transport of biological pigments; proteins involved in skintexture; proteins involved in the fur texture and/or length; andproteins involved in the texture, structural strength, and/or length ofnail, claw, and/or horn; and an inhibitory RNA coding sequence thatinterferes with the expression of a nucleic acid molecule encoding aprotein selected from pigment proteins; proteins involved in thesynthesis and/or transport of biological pigments; proteins involved inskin texture; proteins involved in the fur texture and/or length; andproteins involved in the texture, structural strength, and/or length ofnail, claw, and/or horn.
 2. A non-human transgenic animal comprising acell according to claim
 1. 3. The non-human transgenic animal accordingto claim 2, wherein the inducible gene expression system is selectedfrom a Cre-LoxP recombination system, a FLP-FRT recombination system, atetracycline (Tet)-controlled transcription activation system, anecdysone inducible system, a heat shock on/off system, a lacO/IPTGsystem, a cumate repressor protein CymR system, a nitroreductase system,coumermycin/novobiocin-regulated system, a RheoSwitch Ligand RSL/system,a chimeric bipartite nuclear receptor expression system, a GAL4 system,sterol or steroid or synthetic steroid inducing/repressing system, andany combination thereof.
 4. The non-human transgenic animal according toclaim 3, wherein the inducible gene expression system comprises aCre-LoxP recombination system.
 5. The non-human transgenic animalaccording to claim 4, wherein the Cre-LoxP recombination systemcomprises a lox-stop-lox (LSL) sequence.
 6. The non-human transgenicanimal according to claim 4, wherein the inducing agent is a Crerecombinase protein.
 7. The non-human transgenic animal according toclaim 3, wherein the inducible gene expression system comprises atetracycline (Tet)-controlled transcription activation system.
 8. Thenon-human transgenic animal according to claim 7, wherein the inducingagent is doxycycline.
 9. The non-human transgenic animal according toclaim 2, wherein the promoter is selected from universal promoters,constitutive promoters, tissue-specific promoters, and induciblepromoters.
 10. The non-human transgenic animal according to claim 2,wherein the promoter is selected from cytomegalovirus (CMV) promoter,CMV-chicken beta actin promoter, ubiquitin promoter, JeT promoter, SV40promoter, beta globin promoter, elongation Factor 1 alpha (EF1-alpha)promoter, RSV promoter, Ripply2 promoter, Ticked promoter, Tabbypromoter, Mo-MLV-LTR promoter, Rosa26 promoter, keratinocyte specificpromoters, melanocyte specific promoters, matrix-cell specificpromoters, and dermal papilla-specific promoters.
 11. The non-humantransgenic animal according to claim 2, comprising a cell thatcomprises: a first exogenous nucleic acid molecule operably linked to afirst promoter and under the control of a loxP site, wherein the loxPsite prevents the expression of the first nucleic acid molecule in theabsence of Cre recombinase protein; a second exogenous nucleic acidmolecule encoding a reverse tTA (rtTA), operably linked to a secondpromoter; and a third exogenous nucleic acid molecule encoding a Crerecombinase protein, operably linked to a third promoter under thecontrol of a TetO operator; wherein the first exogenous nucleic acidmolecule is selected from: a nucleic acid molecule encoding a proteinselected from pigment proteins; proteins involved in the synthesisand/or transport of biological pigments; proteins involved in skintexture; proteins involved in the fur texture and/or length; andproteins involved in the texture, structural strength, and/or length ofnail, claw, and/or horn; and an inhibitory RNA coding sequence thatinterferes with the expression of a nucleic acid molecule encoding aprotein selected from pigment proteins; proteins involved in thesynthesis and/or transport of biological pigments; proteins involved inskin texture; proteins involved in the fur texture and/or length; andproteins involved in the texture, structural strength, and/or length ofnail, claw, and/or horn.
 12. The non-human transgenic animal accordingto claim 2, wherein the exogenous nucleic acid molecule encodes aprotein selected from pigment proteins; proteins involved in thesynthesis and/or transport of biological pigments; proteins involved inskin texture; proteins involved in the fur texture and/or length; andproteins involved in the texture, structural strength, and/or length ofnail, claw, and/or horn.
 13. The non-human transgenic animal accordingto claim 12, wherein the exogenous nucleic acid molecule encodes aprotein selected from melanocortin receptor (MC1R), melanocytestimulating hormones (MSH), β-defensin 103, agouti signaling protein(ASP), tyrosinase (TYR), melanocyte-specific transporter protein,Ras-related protein Rab-7, rab protein geranylgeranyltransferasecomponent A2, probable E3 ubiquitin-protein ligase (HERC2), bifunctionalenzyme CarRP-like, lycopene cyclase/phytoene synthase-like, and phytoenedehydrogenase-like.
 14. The non-human transgenic animal according toclaim 2, wherein the exogenous nucleic acid molecule comprises aninhibitory RNA coding sequence that interferes with the expression of anucleic acid molecule encoding a protein selected from pigment proteins;proteins involved in the synthesis and/or transport of biologicalpigments; proteins involved in skin texture; proteins involved in thefur texture and/or length; and proteins involved in the texture,structural strength, and/or length of nail, claw, and/or horn.
 15. Thenon-human transgenic animal according to claim 14, wherein theinhibitory RNA coding sequence interferes with the expression of anucleic acid molecule encoding a keratin protein.
 16. The non-humantransgenic animal according to claim 2, which is a dog, cat, mouse, rat,guinea pig, hamster, horse, cow, pig, sheep, goat, duck, goose, chicken,primate, fish, frog, salamander, snake, lizard, fox, weasel, rabbit,mink, beaver, ermine, otter, sable, seal, coyote, chinchilla, deer,muskrat, or possum.
 17. A method of customizing non-human animal traitscomprising: providing a non-human transgenic animal according to claim2; administering the inducing agent to the transgenic animal therebyinducing the expression of the exogenous nucleic acid molecule.
 18. Themethod according to claim 17, wherein the inducing agent is formulatedwith a carrier for administration to the transgenic animal.
 19. Themethod according to claim 17, wherein the inducible gene expressionsystem comprises a Cre-LoxP recombination system, the inducing agent isa Cre recombinase protein, and the Cre recombinase is topicallyadministered to the transgenic animal.
 20. The method according to claim17, wherein the exogenous nucleic acid molecule is transferred into theanimal via the use of spermatogonial stem cells (SSCs), piggyBac™ mobileDNA technology using transposable elements, Xanthamonas transcriptionactivator-like (TAL) Nucleases (XTNs), or a combination thereof.